Methods of using ajoene for the treatment of tumors

ABSTRACT

The present invention relates to the use of Ajoene to treat integrin-mediated tumors and metastasis.

This application is a division of application number 07/906,850, filedJun. 30, 1992, now abandoned.

BACKGROUND OF THE INVENTION

Ajoene is 4,5,9-trithiadodeca-1,6,11-triene-9-oxide, having a structuralformula as follows: ##STR1##

Ajoene, and a precursor thereof, can be isolated from extracts of garlic(Allium sativum). As the garlic is crushed, alliin in the garlic comesinto contact with allinase in the cell wall to form allicin. Then, inthe presence of a polar molecule such as a lower alcohol or even water,allicin forms Ajoene.

Ajoene has been previously shown to inhibit platelet aggregation byinactivating allosterically the platelet integrin, GP IIb/IIIa(Apitz-Castro R. et al.: Biophys. Res. Commun., 1986, 141:145). Thisinhibition of integrins by Ajoene is reversible.

Also, integrin-mediated formation of cell-to-cell contacts has beenshown to typically be a prerequisite for membrane fusion (Hildreth, J.E. K. and Orentas, R. J., Science, 1989, 244:1075).

DESCRIPTION OF THE INVENTION

By this invention, safe and effective doses of Ajoene may inhibit theprogression of an HIV infection in a patient. Specifically, stereoisomermixtures of Ajoene may be used, although pure (E) and (Z) stereoisomerforms may also be used.

One of the characteristics of an HIV infection so inhibited is theformation of HIV-induced syncytia, in which HIV target cells such aslymphocytes and monocytes clump together to form giant, polynuclearcells in HIV-infected patients. This inhibiting effect would require asufficient dose to provide a concentration of Ajoene of at least 50nanomoles per liter of blood plasma in the patient, and preferably atleast 200 nanomoles of Ajoene per liter of plasma. Transfer of geneticmaterial between cells will, therefore, also be inhibited by Ajoene.

Additionally, it appears that Ajoene inhibits the growth of HIV-infectedcells, HIV, and other viruses, also inhibits the incorporation of HIVinto CD4-negative cells. For this purpose, Ajoene is preferablyadministered in sufficient dose to provide a concentration of at least5,000 nanomoles per liter of patient's blood plasma, although lesserconcentrations may also be effective.

Additionally, the growth of tumors such as T-lymphomas and thedevelopment of melanoma metastasis may be inhibited by administering tothe patient, human or animal, a safe and effective dosage of Ajoene,preferably to provide a concentration of Ajoene to the patient of atleast 50 micromoles per liter of the patient's blood plasma, or at leastin the blood adjacent the tumor, for example in cases of regionalperfusion where steps are taken to minimize the concentration of Ajoeneelsewhere in the body except at the tumor site.

Ajoene serves as an agent that inhibits the aggregation of blood cells,such as platelets and neutrophils. Ajoene may, therefore, exhibitbenefit as an agent for the treatment of pathologies derived fromaggregation of these cells, including, in part, the effects of shock. Incases of shock, platelets and neutrophils aggregate in the blood vesselsof the lungs, which may be a primary cause of death in that oxygentransfer through the lungs is degraded, leading to death. Ajoene maythus counteract at least part of the effects of shock, for example insepsis, anaphylaxis, or in blood loss. The inhibiting effect of Ajoenein this use would typically be the result of a dose to provide aconcentration of Ajoene of at least 10 micromoles per liter of thepatient's blood plasma.

Ajoene can also be administered in effective dosage to suppress manyinflammation processes, for example, those inflammation processes thatoccur in the lungs as a consequence of severe shock. Inflammation is atypical pathological process (i.e., either inherent in or associatedwith a variety of distinct diseases and illnesses), defensive in nature,but potentially dangerous if uncontrolled. There are several indices ofinflammation at the "whole body" level: e.g., hemodynamic disorders(e.g., hyperemia and edema), pain, temperature increment, and functionallesion. At the cellular level, inflammation is characterized byleukocyte emigration into tissues (where they phagocytose bacteria,viruses, or other invaders) and platelet aggregation (to localize theinfection), which emigration and aggregation may also have a destructiveeffect on tissue. At the molecular level, inflammation is characterizedby activation of at least three plasma defense systems (complement,kinin, and coagulation cascades), and by synthesis of cytokines andeicosanoids.

When inflammation is generalized, as in the case of shock, the variousindices of inflammation may become disseminated and occur throughout theentire organismn.

Without wishing to be limited to any particular mechanism of operationof Ajoene, it is believed that the beneficial effects of Ajoene areachieved because Ajoene is an inactivating agent for several integrinreceptors of cells. Thus, it is believed that other inactivating agentsfor cell integrin receptors may exhibit similar benefits as Ajoene,particularly with respect to the suppression of adhesion eventsmediating HIV spread in the body (e.g., adhesion-induced HIV budding,infection of CD4⁻ cells, cell-to-cell transmission of infection by cellfusion, infection by virus having adhesion molecules captured from hostcells, syncytia formation in the central nervous system).

Work by others has shown that HIV-1 Tat protein stimulates the growth ofAIDS-Kaposils sarcoma (AIDS-KS) cells by interacting with the integrinsα₅ β₁ and α_(v) β₃. Ajoene should, therefore, be effective in treatingKaposi's sarcoma in view of its ability to inactivate integrins.

The above disclosure and the examples below are for illustrativepurposes only, and are not intended to limit the invention of thisapplication, which is as defined in the claims below.

EXAMPLE 1 Ajoene as an Inhibitor of HIV-Mediated Syncytia Formation

Intact H9 cells and chronically infected H9:RF cells (a clonedpopulation of H9 cells harboring HIV genome) were maintained insuspension in RPMI-1640 medium supplemented with 10% (v/v)heat-inactivated fetal calf serum, 50 micrograms/ml gentamycin and 2 mML-glutamine. Syncytium formation was assessed by co-cultivation of H9cells and H9:RF cells (2:1) for 16 hrs with various concentrations ofAjoene in 96-well flat-bottomed plates, 3×10⁵ cells per well. By the endof the incubation period syncytia were counted. IC₅₀ for Ajoene wascalculated by plotting relative amount of syncytia (percent of thesyncytia formed in the absence of the compound) against Ajoeneconcentration. If not specified otherwise, Ajoene was used as a 1:1mixture of (E)- and(Z)-stereoisomers.

Specifically, Ajoene inhibited fusion of intact H9 cells withHIV-infected H9:RF cells (IC₅₀ of 50 nanomoles per liter Ajoeneconcentration at 16 hrs incubation).

EXAMPLE 2 Ajoene as an Inactivator of Several Distinct IntegrinReceptors

Platelet aggregation was measured turbidimetrically on a standardplatelet aggregometer equipped with a recorder. Samples of platelet-richplasma (PRP) were standardized with respect to the cell density (3×10⁸per ml); maximal light transmission (LT) was calibrated withplatelet-poor plasma (PPP). Aliquots of PRP (0.25 ml in glass cuvettes)were stirred in the cell of the device (1000 rpm, 37° C.) with Ajoene(0-100 micromoles per liter final concentration) for 1 min., followed byaddition of the stimuli. LT increments were traced for 5 min. Maximalrate of aggregation was derived from the slope of each curve; IC₅₀ forAjoene was determined by a method such as that described in VrzheshchPV, Tatarintsev AV, Yershov DE et al.: Cell Response Kinetics: thePhenomenon of Supercooperativity in Aggregation of Human Platelets.Dokl. Akad. Nauk SSSR, 1989, 307:477 (in Russian). Neutrophilaggregation was assayed as described above for the platelet system, withsome modifications. Freshly isolated neutrophils were finally suspendedin a buffer (120 mM NaCl, 4 mM KCl, 10 mM dextrose, 2 mM CaCl₂.2H₂ O, 2mM MgCl₂.6H₂ O, and 20 mM Tris; pH 7.4) to the final density of 107cells per ml. Aliquots of neutrophil suspension (0.25 ml in siliconizedglass cuvettes; 10⁷ cells per ml) were stirred in the cell of theaggregometer with 5 micrograms per ml cytochalasin B (1000 rpm, 37° C.)for 1 min. Ajoene (0-50 micromoles per liter) andN-formyl-L-methionyl-L-leucyl-L-phenylalanine (10⁻⁷ M) were thenintroduced into the suspension sequentially, with an interval of 1 min.Maximum LT (100%) was calibrated with the same buffer. H9 and Jurkatcells were cultured as described above in Example 1.

Ajoene inhibited platelet aggregation in PRP with an IC₅₀ of about 50micromoles per liter. Ajoene blockedN-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated neutrophilaggregation (IC₁₀₀ of about 10 micromoles per liter) and caused rapiddeaggregation when added to aggregated neutrophils. The clustersnormally formed by H9 and Jurkat cells in culture were disrupted byAjoene with an IC₅₀ of about 50 nanomoles per liter.

Though the requirement of integrins for contact interactions in eitherof these systems has long been established, the molecular mechanisms areclearly nonidentical. It is known that in platelets the relevantintegrin is GP IIb/IIIa. Neutrophils form contacts via interaction ofLFA-1 and/or Mac-1 with their surface counterstructures, while cohesionof T-cells is supported by LFA-1 and by fibronectin-binding VLAintegrins. Hence, Ajoene is apparently capable of inactivating integrinreceptors of at least three distinct subfamilies (β₁, β₂ and β₃) Oneprobable mechanism is that the compound either binds to a region whichis highly conserved within the members of the integrin superfamily, orsomehow alters the microenvironment, thereby inactivating the wholereceptor. At the cellular level this inactivation appears as a severe(but controllable) deficiency in the respective cell-specific integrins,i.e., inability for aggregation (platelets, neutrophils), disintegrationof cell clusters (intact H9 and Jurkat cells), failure to fuse(HIV-infected cells; Example 1).

EXAMPLE 3 Ajoene As An Inhibitor of HIV Replication

LAV-BRU 1 isolate of HIV-1 and RF isolate of HIV-1 were propagated inCEM and H9 cells, respectively. A 20-ml log-phase culture containing7×10⁵ cells per ml was incubated at 37° C. for 24 hrs. Cultures withless than 10% trypan blue stained cells were used as a source of virus.To harvest HIV particles, producer cells were pelleted by centrifugation(400 g, 5 min) and the supernatant containing HIV-1 particles wasfiltered through a 0.45 micrometer Millipore filter, aliquoted andstored at -80° C. The titer of HIV-1 ranged from 1×10⁵ to 2×10⁵ CCID₅₀(50% cell culture infective dose). To determine whether Ajoene couldalso influence HIV replication, CEM13 and H9 cells were inoculated withappropriate amounts of HIV (LAV-BRU 1 and RF, respectively), to give anm.o.i. (multeity of infection) of 0.1 in either experimental setting.Ajoene (or its vehicle) was added to 10⁷ infected cells resuspended inthe growth medium and the cells were plated in 96-well flat-bottomedplates. After 72 hrs the bulk of HIV antigens was measured by solidphase immunoassay as described in Zhdanov VM, Karamov EV, Arakelov SA,et al.: An Enzyme Immunoassay for Detection of Antigen and Antibody toHuman Immunodeficiency Virus and the Use Thereof For serological Surveyof Different Population Groups. Vop Virusol 1988, N3:292 (in Russian).In a series of separate experiments Ajoene was introduced into platedcell cultures stepwise, i.e. in aliquots of 50 nanomoles per literconcentration per 12 hrs of incubation, the first addition coincidingwith the moment of inoculation.

Replication of HIV-1 (RF) in H9 cells was inhibited with an IC₅₀ of 25micromoles per liter (m.o.i. 0.1; 72 hrs of incubation). Assessment ofHIV-1 (LAV-BRU 1) replication in CEM13 cells under the same experimentalconditions revealed a more pronounced anti-viral activity (IC₅₀ of about5 micromoles per liter concentration). A considerable increase in thelatter became evident when the concentration of the compound wasincreased stepwise (50 nanomoles per liter concentration per 12 hrs ofincubation; CEM13--LAV-BRU 1 system; inhibition by 30%; totalconcentration 0.25 micromoles per liter; m.o.i. 0.1; 72 hrs ofincubation).

EXAMPLE 4 Ajoene as a Potential Anti-Shock Agent

Neutrophil aggregation within the lung microvasculature is known to be akey event in the development of the so-called adult respiratory distresssyndrome (ARDS), the main cause of death in patients suffering shock.Also, administration of neutrophil-aggregating agents (such asphorbol-myristate-acetate, complement anaphylotoxins orN-formyl-L-methionyl-L-phenylalanine, calcium ionophore A23187,arachidonic acid, platelet-activating factor) to laboratory animals alsoresults in the development of ARDS. Some inhibitors of neutrophilaggregation, such as antagonists of the platelet-activating factor, havebeen shown to prevent lethality in experimental septic shock, ToyofokuT. et al.: Effects of ONO-6340, a Platelet-Activating Factor Antagonist,on Endotoxin Shock in Unanesthetized Sheep. Prostaglandins, 1986,31:271). It is believed that beneficial effects of glucocorticoids invarious shock states are largely due to their ability for inhibition ofneutrophil aggregation (Hammerschmidt, et al., J. Clin. Invest. 1979,63:798; Cronstein, B. N. et al., Proc. Natl. Acad. Sci. USA, 1992, 89:9991-99).

The antiaggregatory activity of Ajoene disclosed in Example 2 above issufficiently high to expect that Ajoene will be effective both for theprevention and treatment of various shock states by suppressing suchaggregation.

EXAMPLE 5 Ajoene as an Antineoplastic Agent

Leukemic (CEM, MT2, MT4) and lymphoid (H9, Jurkat) T-cells were culturedas described above (Example 1) with various concentrations of Ajoene,and the toxic effects were assessed by the cytocidal activity (LD₁₀₀).

For T-lymphoid tumor cells, LD₁₀₀ was achieved after 72 hrs of culturingin a media of about 50 micromoles of Ajoene per liter.

For Leukemia T cells LD₁₀₀ was achieved after 72 hrs of culturing in amedia of about 500 micromoles of Ajoene per liter.

EXAMPLE 6 Ajoene as an Antimetastatic Agent

Male C57BL/6 mice were used as experimental animals. Murine plateletaggregation was measured as described above (Example 3). To evaluate theeffect of the compound on experimental metastasis, a suspension of 10⁵melanoma B16 cells was pretreated for 30 min with Ajoene or its vehicle,and the suspension (including Ajoene or its vehicle) was injectedintravenously into mice. The mice were sacrificed 3 weekspost-injection, and their lungs subjected to microscopic examination. Toevaluate the effect on tumor cell implantation, mice were inoculatedsubcutaneously with 10⁶ B16 cells (pretreated for 15 min with Ajoene orits vehicle) in their pretreatment medium, and the volume of the tumorsformed was measured 14 and 21 days after the injection.

Ajoene inhibited ADP-induced aggregation of PRP with an IC₁₀₀ of 200micromoles per liter. Pretreatment of B16 cells with Ajoene (200micromoles per liter) rendered them incapable of colonizing lungs.Subcutaneous implantation was prevented by exposing the cells to 2500micromolar concentration of Ajoene. At 2.5 micromolar concentration ofthe compound, the volume of melanomas was reduced by one-half.

EXAMPLE 7 Integrin-Inactivating Properties of Ajoene are notStereospecific

Synthetic stereoisomers of Ajoene exhibited equal antiaggregatoryactivity (IC₅₀ of about 50 micromolar concentration of Ajoene forplatelets, IC₁₀₀ of about 10 micromolar concentration of Ajoene forneutrophils).

BRIEF DESCRIPTION OF THE DRAWING

Referring to FIG. 1, the effect of Ajoene on syncytia formation and HIVreplication is shown.

For curve 1, the effect of Ajoene on the infusion of cultured, intact H9cells with HIV infected H9:RF cells is disclosed. For this curve, thevertical graph axis pertains to the maximum amount of syncytia formed inthe absence of Ajoene (100 percent), while the points on line 1represent percentages of such an amount of syncytia formed in thepresence of varying concentrations of Ajoene (micromoles per liter). TheIC₅₀ of syncytia formation was found at a concentration of 0.045micromole of Ajoene per liter. Essentially no syncytia were found at aconcentration of 50 micromoles of Ajoene per liter.

Referring to curve 2, the antiviral activity of Ajoene is shown, asassessed by the inhibition of HIV-1 (Lav--BRU 1) replication in culturedCEM13 cells. Here, the vertical axis represents the percentage of HIVantigens detected by solid phase immunoassay (previously mentioned) inthe absence of Ajoene.

The IC₅₀ was achieved under these conditions at about a 5 micromoleconcentration of Ajoene.

Thus, Ajoene exhibits significant potential as an effective anti-HIVdrug to reduce or eliminate the course of the disease while exhibitingrelatively low toxic effects. Ajoene is also effective against manytumor cells and their metastasis. In addition, Ajoene exhibitssignificant potential as an effective drug to block cell aggregation,for example in various shock states. Finally, Ajoene may be used totreat inflammation.

To treat one of these conditions, an effective amount of Ajoene isadministered. Effective dosage forms, modes of administration and dosageamounts, may be determined empirically, and making such determinationsis within the skill of the art. It is understood by those skilled in theart that the dosage amount will vary with the severity of the condition,the route of administration, the rate of excretion, the duration of thetreatment, the identity of any other drugs being administered, the age,size and species of the animal, and like factors well known in themedical and veterinary arts. In general, a suitable daily dose of Ajoenewill be that amount of the compound which is the lowest dose effectiveto produce a therapeutic effect. However, the total daily dosage will bedetermined by an attending physician or veterinarian within the scope ofsound medical judgment. If desired, the effective daily dose of Ajoenemay be administered as two, three, four, five, six or more sub-doses,administered separately at appropriate intervals throughout the day.

Ajoene may be administered in any desired and effective manner: as apharmaceutical preparation for oral ingestion, or for parenteral orother administration in any appropriate manner such as intraperitoneal,subcutaneous, topical, inhalation, intrapulmonary, rectal, vaginal,sublingual, intramuscular, intravenous, intraarterial, intrathecal, orintralymphatic. For instance, the topical application of Ajoene tomucous membranes can be used to prevent HIV infection of mucosal cells,an important route of HIV transmission. In addition, intralymphaticadministration of Ajoene may be advantageous in preventing the spread ofHIV within the body. Further, Ajoene may be administered in conjunctionwith other anti-HIV drugs, other chemotherapy agents for tumor ormetastasis treatment, and other anti-shock or anti-inflammation drugs ortreatments. The Ajoene may be encapsulated or otherwise protectedagainst gastric or other secretions, if desired.

The above has been offered for illustrative purposes only, and is notintended to limit the scope of the invention of this application, whichis as described in the claims below.

I claim:
 1. A method of inhibiting integrin-mediated tumor metastasis inan animal in need thereof comprising administering to the animal anamount of ajoene effective to inhibit integrins and metastasis of thetumor.
 2. The method of claim 1 wherein the ajoene is administered insufficient amount to provide a concentration of at least 50 micromolesper liter of blood plasma.
 3. The method of claim 2 wherein the ajoeneis administered in sufficient amount to provide a concentration of atleast 200 micromoles per liter of blood plasma.
 4. The method of claim 1or wherein the agent is administered intralymphatically.
 5. A method oftreating an integrin-mediated cancer in an animal in need thereofcomprising administering to the animal an amount of ajoene effective toinhibit integrins and the cancer.
 6. The method of claim 5 wherein thecancer is a T-cell cancer.
 7. The method of claim 5 wherein the canceris a melanoma.
 8. The method of claim 5, 6 or 7 wherein the ajoene isadministered in sufficient amount to provide a concentration of at least50 micromoles per liter of blood plasma.
 9. The method of claim 8wherein the ajoene is administered in sufficient amount to provide aconcentration of at least 2500 micromoles per liter of blood plasma. 10.The method of claim 5, 6 or 7 wherein the ajoene is administeredintralymphatically.
 11. A method of treating Kaposi's sarcoma in anHIV-infected patient comprising administering to the patient atherapeutically effective amount of ajoene.